Abstract: ObjectiveSCoT-PCR reaction system of Pueraria DC.was optimized and SCoT primers that suitable for Pueraria were screened to provide reference for using SCoT molecular marker in Pueraria germplasm resources identifi-cation,genetic diversity and molecular mark assisted breeding.MethodThis study carried out orthogonal design to opti-mize five factors including DNA template amount,dNTPs concentration,Mg2+concentration,Ta q DNA polymerase amount and primer concentration.SCoT primers that suitable for Pueraria were screened out by the optimal SCoT-PCR re-action system,and this system was testified later.ResultThe results showed that,the optimized SCoT-PCR reaction sys-tem(20.00μL)for Pueraria was composed of 50.00 ng DNA template,0.25 mmol/L dNTPs,1.50 mmol/L Mg2+,0.80 μmol/L primer and 0.50 U Ta q DNA polymerase.Out of 36 primers,35 SCoT primers were selected which could ampli-fied clear bands from Pueraria based on the optimized reaction system.Using three primers to amplify 18 Puerariae mate-rials,the PCR products showed fine repeatability,stability and polymorphism.ConclusionThe optimal SCoT-PCR reac-tion system for Pueraria obtained and 35 selected SCoT primers can be used for germplasm resources identification,genetic diversity analysis and molecular marker development.